Growth arrest homeobox gene

ABSTRACT

A novel growth arrest homeobox gene has been discovered and the nucleotide sequences have been determined in both the rat and the human. The expression of the novel homeobox gene inhibits vascular smooth muscle cell growth. The growth arrest homeobox gene hereinafter referred to as the “Gax gene” and its corresponding proteins are useful in the study of vascular smooth muscle cell proliferation and in the treatment of blood vessel diseases that result from excessive smooth muscle cell proliferation, particularly after balloon angioplasty.

BACKGROUND OF THE INVENTION

The leading cause of death in the United States and in most developedcountries, is atherosclerosis. Atherosclerosis is a disease affectingthe large and medium size muscular arteries such as the coronary orcarotid arteries and the large elastic arteries such as the aorta,iliac, and femoral arteries. This disease causes narrowing andcalcification of arteries. The narrowing results from deposits ofsubstances in the blood in combination with proliferating vascularsmooth muscle cells.

The deposits known as atherosclerotic plaques are comprised oflipoproteins, mainly cholesterol, proliferating vascular smooth musclecells and fibrous tissue, and extra cellular matrix components, whichare secreted by vascular smooth muscle cells. As the plaques grow, theynarrow the lumen of the vessel decreasing arterial blood flow andweakening the effected arteries. The resulting complications potentiallyinclude a complete blockage of the lumen of the artery, with ischemiaand necrosis of the organ supplied by the artery, ulceration andthrombus formation with associated embolism, calcification, andaneurysmal dilation. When atherosclerosis causes occlusion of thecoronary arteries, it leads to myocardial disfunction, ischemia andinfarction and often death. Indeed, 20-25% of deaths in the UnitedStates are attributable to atherosclerotic heart disease.Atherosclerosis also leads to lower extremity gangrene, strokes,mesenteric occlusion, ischemic encephalopathy, and renal failure,depending on the specific vasculature involved. Approximately 50% of alldeaths in the United States can be attributed to atherosclerosis and itscomplications.

Present treatments for atherosclerosis include drugs and surgery,including ballon angioplasty. As a result of angioplasty, vascularsmooth muscle cells de-differentiate and proliferate and leading toleading to reocclusion of the vessel. These de-differentiated vascularsmooth muscle cells deposit collagen and other matrix substances, thatcontribute to the narrowing of vessel. Vascular cells secrete growthfactors such as platelet derived growth factor, which induces bothchemotaxis and proliferation of vascular smooth muscle cells.

Many of the present drug therapies treat a predisposing condition suchas hyperlipidemia, hypertension, and hypercholesterolemia, in an attemptto slow or halt the progression of the disease. Other drug therapies areaimed at preventing platelet aggregation or the coagulation cascade.Unfortunately, the drug treatments do not reverse existing conditions.

Surgical treatments include coronary artery bypass grafting, balloonangioplasty, or vessel endarterectomy which, when successful, bypass orunblock occluded arteries thereby restoring blood flow through theartery. The surgical treatments do not halt or reverse the progressionof the disease because they do not affect smooth muscle cellproliferation and secretion of extra cellular matrix components.

The bypass surgeries, particularly the coronary bypass surgeries, aremajor, complicated surgeries which involve a significant degree of risk.The balloon angioplasty, while also a surgical procedure, is less risky.In balloon angioplasty, a catheter having a deflated balloon is insertedinto an artery and positioned next to the plaque. The balloon isinflated thereby compressing the plaque against the arterial wall. As aresult, the occlusion is decreased and increased blood flow is restored.However, the balloon angioplasty injures the arterial wall. As a result,the underlying vascular smooth muscle cells migrate to the intima, andsynthesize and excrete extracellular matrix components eventuallyleading to the reocclusion of the vessel. Of the estimated 400,000coronary artery balloon angioplasties performed each year in the UnitedStates, 40% fail due to reocclusion requiring a repeat procedure orcoronary bypass surgery. Bypass surgeries also have a significant rateof failure due to internal hyperplasia, which involves excessiveproliferation of vascular smooth muscle cells at the sites of vascularanastamoses.

Attempts have been made to prevent reocclusion of vessels after balloonangioplasties in experimental animals. One approach has been to treatrat carotid arteries with antisense oligonucleotides directed againstthe c-myb gene following balloon angioplasty de-endothelialization. Invascular smooth muscle cells the The expression of the c-myb gene isup-regulated during the G1 to S transition of the cell cycle, and theactivation of c-myb expression is required for further cell cycleprogression. The antisense oligonucleotides to c-myb blocked smoothmuscle cell proliferation following balloon angioplasty. However, theantisense oligonucleotides are applied in a pleuronic gel to theadventitia, that is, the exterior, rather than the lumen side of theaffected vessel. Exposing the the exterior of the vessel requiresadditional surgery with its attendant risks, and is therefore notdesirable.

It would be desirable to have a nonsurgical treatment, used inconjunction with balloon angioplasties to reduce vascular smooth musclecell proliferation.

SUMMARY OF THE INVENTION

A novel growth arrest homeobox gene has been discovered and thenucleotide sequences have been determined in both the rat and the human.The expression of the novel homeobox gene inhibits vascular smoothmuscle cell growth. The growth arrest homeobox gene hereinafter referredto as the “Gax gene” and its corresponding proteins are useful in thestudy of vascular smooth muscle cell proliferation and in the treatmentof blood vessel diseases that result from excessive smooth muscle cellproliferation, particularly after balloon angioplasty.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of rat Gax gene with the predictedamino acid sequence listed below the nucleotide sequence. The homeoboxis indicated by a box, and the CAX nucleotide repeat, where X is ethercytosine or guanine, is underlined. A polyadenylation signal is inboldface and italics. Putative consensus sites are indicated as follows:for phosphorylation by protein kinase C, circles; for cyclic AMP(cAMP)-dependent protein kinase, squares; for casein kinase II,diamonds; and for histone H1 kinase, triangles. Residues which couldpotentially be a target for either cAMP-dependent protein kinase orprotein kinase C are both circled and boxed.

FIG. 2 is the map of mouse chromosome 12 showing the location of the Gaxgene;

FIG. 3 is the nucleotide sequence of human Gax gene with the predictedamino acid sequence listed below the nucleotide sequence;

FIG. 4 is a map of human Gax gene showing how the separately clonedfragments were joined and oriented in the plasmid, Bluescript IISK+;

FIG. 5A is a northern blot showing Gax RNA levels in vascular smoothmuscle cells in response to 10% fetal calf serum after 4, 24, and 48hours; lane Q is RNA from quiescent cells; GAPDH is rat glyceraldehyde3-phosphate dehydrogenase;

FIG. 5B is a northern blot showing Gax RNA levels and Hox 1.3 RNA levelsin vascular smooth muscle cells in response to 10 ng/ml human plateletderived growth factor at 0.25, 0.5, 1, 2, and 4 hours, lane Q is RNAfrom quiescent vascular smooth muscle cells;

FIG. 6 is a graph of changes in relative Gax mRNA levels in vascularsmooth muscle cells in response to 10% fetal calf serum and 10 mg/ml ofthe PDGF isoforms; the circles represent PDGF-AA, the squares representPDGF-BB, the diamonds represent fetal calf serum, and the trianglesrepresent PDGF-AB;

FIG. 7 is a graph showing ³H-thymidine uptake in vascular smooth musclecells at various times after stimulation with fetal calf serum and PDGFisoforms; the circles represent PDGF-AA, the triangles representPDGF-AB, the squares represent PDGF-BB, the diamonds represent fetalcalf serum, and the solid squares represent no mitogen;

FIG. 8 is a graph showing relative Gax mRNA levels in vascular smoothmuscle cells in response to varying doses of PDGF-AB, represented bytriangles, and PDGF-BB, represented by squares;

FIG. 9 is a graph showing relative Gax mRNA levels in vascular smoothmuscle cells in response to varying doses of fetal calf serum;

FIG. 10 is a graph showing relative Gax mRNA levels in vascular smoothmuscle cells in response to fetal calf serum withdrawal;

FIG. 11 is a dose response curve showing % inhibition of growth invascular smooth muscle cells in response to varying doses ofmicroinjected GST-Gax protein;

FIG. 12 is a graph showing percent inhibition of mitogen induced DNAsynthesis in vascular smooth muscle cells in response to: ras (Leu-61)protein; ras (Leu-61) protein in combination with the GST-Gax protein;GST-Gax protein; and the GST;

FIG. 13 is a graph showing percent inhibition of vascular smooth musclecell entry into S phase by microinjected GST-Gax protein over time andthe ³H-thymidine uptake over the same time period;

FIG. 14 is a graph showing the ratio of the Gax mRNA toglyceraldehyde-3-phosphate dehydrogenase designated “G3” level fromnormal vascular tissue and times following acute blood vessel injury.

DETAILED DESCRIPTION OF THE INVENTION

A novel gene, the Gax gene, has been discovered, the expression of whichinhibits vascular smooth muscle cell growth. The Gax gene and theprotein it encodes, referred to herein as the “Gax protein” are usefulin the study of vascular smooth muscle cell proliferation and ininhibiting smooth muscle cell proliferation. The inhibition of vascularsmooth muscle cell proliferation, particularly by genetic therapy, isalso useful in the treatment of vascular diseases associated withexcessive smooth muscle cell proliferation.

Nucleotide sequences, such as the Gax gene or portions therof, or mRNAare administered to the vascular cells, preferably during a balloonangioplasty procedure, to inhibit the proliferation of vascular smoothmuscle cells. The nucleotide sequences are delivered, preferably to theinterior of the vessel wall during balloon angioplasty procedurepreferably by a perforated balloon catheter. Genes are transfered fromvectors into vascular smooth muscle cells in vivo where the genes areexpressed. Suitable vectors and procedures for the transfer ofnucleotides are found in

Nabel, E. G., et al. “Site-Specific Gene Expression in Vivo by DirectGene Transfer into the Arterial Wall” (1990) Science Vol. 249, pp.1285-1288, which is incorporated herein by reference. Specializedperforated balloon catheters which deliver nucleotide sequences tovessel walls employing viral and non-viral vectors are used for deliveryof nucleotide sequences and a description of the catheter's structureand use may be found in Flugelman M. Y., et al. “Low Level In Vivo GeneTransfer Into the Arterial Wall Through a Perforated Balloon Catheter”Circulation, Vol. 85, No. 3, pp. 1110-1117 (March 1992) which isincorporated herein by reference.

Genetic therapy, preferably by the over expression of the Gax gene,restores the proliferating vascular smooth muscle cells to a more normalphenotype, thus preventing or reducing the smooth muscle proliferationthat is associated with the formation of the atheromatous plaque andwith internal arterial thickening following balloon angioplasty. Inaddition to preventing or reducing the reocclusion of the vessel, suchgenetic therapy decreases the risks associated with additionalsurgeries. Also, the Gax proteins or portions thereof, are administeredto vascular cells preferably employing the perforated catheter, toinhibit the proliferation of vascular smooth muscle cells.

The molecular control of cellular proliferation is not well understood.A class of genes, known as Homeobox genes, encode a class oftranscription factors which are important in embryogenesis, tissuespecific gene expression and cell differentiation. The homeobox genesshare a highly conserved 183 nucletide sequence that is referred to asthe “homeobox”. The homeobox encodes a 61 amino acid helix-turn-helixmotif that binds to adenine and thymine rich gene regulatory sequenceswith high affinity. Several vertebrate homeobox proteins have been shownto be transcription factors required for expression of lineage-specificgenes. The tissue-specific transcription factors bind to DNA and repressor induce groups of subordinate genes. Many, but not all of thesehomeobox genes are located in one of four major clusters known as Hoxclusters, designated Hox-1, Hox-2, Hox-3 and Hox-4. The Hox genes areexpressed in the developing embryo, in distinct overlapping spatialpatterns along the anterior-posterior axis which parallels the Hox geneorder along the chromosome. Homeobox transcription factors control axialpatterning, cell migration and differentiation in the developing embryoand are involved in the maintenance of tissue specific gene expressionin adult organisms.

A new homebox gene has been discovered, isolated and sequenced in boththe rat and human. This new gene is a growth arrest specific homeoboxgene and is referred to herein as the “Gax gene”. The expression of theGax gene is restricted to the cardiovascular system, and in particular,to vascular smooth muscle cells where it functions as a negativeregulator of cell proliferation.

Isolation of the Rat Gax cDNA

An adult rat aorta cDNA library in λ ZAP, from Stratagene, was screenedwith a 64-fold degenerate 29-mer oligonucleotide containing threeinosine residues directed at the most highly conserved region of theantennapedia homeodomain (helix 3), with the following sequence, where Irepresents inosine: 5′-AA(A/G)ATITGGTT(T/C)CA(A/G)AA(C/T) (A/C)GI(A/C)GIATGAA-3′.

Recombinant phage colonies in Escherichia coli were adsorbed induplicate to nitrocellulose membranes and hybridized at 42° C. with thisoligonucleotide end labeled with (T-³²P)ATP in a mixture containing 0.5M sodium phosphate at pH 7.0, 7% sodium dodecyl sulfate, 1 mM EDTA, and1% bovine serum albumin. The filters were washed with a final stringencyof 0.5×SSC (1×SSC in 150 mM NaCl with 15 mM sodium citrate at pH7.0)-0.1% sodium dodecyl sulfate at 42° C. and exposed to X-ray film.Thirteen positive signals were isolated and rescreened until the cloneswere plaque purified. The plasmids containing the clones in λ ZAP vectorwere then excised by the protocol recommended by the manufacturer andsequenced on both strands with sequenase version 2.0 from United StatesBiochemicals. From 500,000 plaques, 13 positive clones were isolated, 12of which contained homeodomains. Nine of the isolated clones werederived from previously described homeobox genes: Hox-1.3, Hox-1.4,Hox-1.11, and rat homeobox R1b. However, three clones represented thecDNA designated herein as the “Gax” gene. Homology searches wereperformed via the GenBank and EMBL data bases, version 73, by using theBLAST algorithm (4).

Nucleotide Sequence of the Rat Gax Gene

The nucleotide sequence of the rat Gax gene is shown in FIG. 1. The cDNAencoding Gax is 2,244 base pairs in length, which corresponds to thesize of the Gax transcript, that is the Gax mRNA, which is about 2.3 to2.4 kb as determined by Northern blot analysis. The Gax cDNA has an openreading frame from nucleotide residues 197 to 1108 beginning with anin-frame methionine that conforms to the eukaryotic consensus sequencefor the start of translation and is preceded by multiple stop codons inall three reading frames. The open reading frame of the cDNA predicts a33.6-kDa protein containing 303 amino acids with a homeodomain fromamino acid residues 185 to 245, as shown in FIG. 1. To confirm that thiscDNA was capable of producing a protein product, the Gax open readingframe was fused in frame to the pQE-9 E. coli expression vector, fromQiagen, Inc., Chatsworth, Calif. and expressed in bacteria according toHochuli, E., et. al. (1988) “Genetic Approach to Facilitate Purificationof Recombinant Proteins with a Novel Metal Chelate Adsorbent”Bio/Technology Vol. 6, pp. 1321-1325. E. coli containing this plasmidexpressed a new phosphorylated protein of about 30 to about 36 kDa asdetermined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and extracts from these E. coli cells displayed a weak binding activityfor the adenine and thymine rich, MHox-binding site in the creatinekinase M enhancer.

The cDNA encoding the rat Gax gene also contains a long 3′-untranslatedregion, from bases 1109 to 2244, with a polyadenylation signal at base2237, as shown in FIG. 1. The region between amino acids 87 and 184contains 23 serine amino acids out of 88 amino acids and 10 prolineamino acids out of 88 amino acids and contains several consensussequences for phosphorylation by protein kinases. Gax also possesses astructural feature which is also found in several transcription factors,including homeodomain proteins, known as the CAX or Opa transcribedrepeat. The Opa transcribed repeat encodes a stretch of glutamines andhistidines; in the rat Gax gene it encodes 18 residues, of which 12 areconsecutive histidines. This motif is shared by other transcriptionfactors, such as the zinc finger gene YY-1, as well as by severalhomeobox genes, including H2.0, HB24, ERA-1 (Hox-1.6), Dual bar, andTes-1. The Gax protein may require post-translational modifications forfull activity, modifications that bacterially produced proteins do notundergo. Since the Gax protein has multiple consensus sites forphosphorylation by protein kinases, it is possible that its activity isactivated or otherwise modulated by phosphorylation at one or more ofthose sites.

The Gax Gene Maps to a Chromosome 12 of the Mouse Genome

Gax is located on chromosome 12 as shown in FIG. 2 of the mouse and isnot a part of the Hox-1, Hox-2, Hox-3, or Hox-4 gene clusters, which arelocated on chromosomes 6, 11, 15, and 2, respectively, McGinnis, W., andR. Krumlauf, (1992) “Homeobox genes and Axial Patterning” Cell, Vol. 68,pp. 283-302. Also Gax does not cosegregate with any other homeobox genespreviously mapped in the interspecific backcross. A comparison was doneof the interspecific map of chromosome 12 with a composite mouse linkagemap that reports the map location of many uncloned mouse mutations usingGBASE, a computerized data base maintained at The Jackson Laboratory,Bar Harbor, Me. The Gax gene mapped in a region of the composite mapthat lacks mouse mutations with a phenotype that might be expected foran alteration in this locus.

The mouse chromosomal location of the Gax was determined byinterspecific backcross analysis using progeny generated by mating(C57BL/6J×Mus spretus)F₁ females and C57BL/6J males. The C57BL/6J and M.spretus DNAs were digested with several enzymes and analyzed by Southernblot hybridization for informative restriction fragment lengthpolymorphisms with a rat cDNA Gax probe. The probe, a 1,155-bp rat cDNAclone, was labeled with (α-³²P)dCTP by using a random prime labeling kitfrom Amersham and washing was done with a final stringency of 0.2×SSCP(34)-0.1% sodium dodecyl sulfate, 65° C. A major fragment of 4.2 kb wasdetected in HincII-digested C57BL/6J DNA, and major fragments of 3.6 and2.7 kb were detected in HincII-digested M. spretus DNA. The 3.6-kb and2.7-kb M. spretus HincII restriction fragment length polymorphisms wereused to monitor the segregation of the Gax locus in backcross mice.Recombination distances were calculated by using the computer programSPRETUS MADNESS. Gene order was determined by minimizing the number ofrecombination events required to explain the allele distributionpatterns.

The mapping results indicated that the mouse Gax gene is located in theproximal region of mouse chromosome 12 linked to neuroblastomamyc-related oncogene 1 (Nmyc-1), the laminin B1 subunit gene (Lamb-1), aDNA segment, chromosome 12, the Nyu 1 gene (D12Nyu1), and the β-spectringene (Spnb-1). The ratios of the total number of mice exhibitingrecombinant chromosomes to the total number of mice analyzed for eachpair of loci and the most likely gene order are as follows:centromere-Nmyc-1-19/193-Lamb-1-9/166-Gax-10/166-D12Nyu1-19/185-Spnb-1.The recombination frequencies, expressed as genetic distances incentimorgans ± the standard error, are as follows:Nmyc-1-9.8±2.2-Lamb-1-5.4±1.8-Gax-6.0±1.9-D12Nyu1-10.3±2.2-Spnb-1.

Gax Gene Expression in Rat Tissue

It has been discovered that the Gax transcript is largely confined tothe cardiovascular system, including the descending thoracic aorta,where it is expressed at higher levels than in other tissues, and theheart. Gax gene expression was also detected in the adult lung andkidney where it is found in mesangial cells. No Gax gene expression wasdetected in the brain, liver, skeletal muscle, spleen, stomach, ortestes, nor was expression detected in the intestine or pancreas. Incontrast, the Gax gene was more widely expressed in the developingembryo, with the transcript detectable in the developing cardiovascularsystem, multiple mesodermal tissues, and some ectodermal tissues.

The 2.3-kb to 2.4-kb Gax RNA transcript was detected in smooth musclecells cultured from adult rat aorta, consistent with the in situhybridization findings and the fact that Gax was originally isolatedfrom a vascular smooth muscle library. The Gax transcript was alsodetected in rat vascular smooth muscle cells transformed by simian virus40. However, no Gax gene expression was detected in either of two celllines derived from embryonic rat aortic smooth muscle, A7r5 and A10. TheGax transcript was also not detected in NIH 3T3 fibroblasts, or humanforeskin fibroblasts. The Gax transcript was not detected in theskeletal muscle cell line C2C12. A relatively high level of Gax geneexpression was detected in cultured rat mesangial cells. Mesangial cellsshare many similarities to vascular smooth muscle cells, bothstructurally and functionally, and proliferate abnormally in renaldiseases such as glomerulonephritis and glomerulosclerosis.

Isolation of the Human Gax cDNA

The nucleotide sequence of the human Gax gene coding sequence is shownin FIG. 3. Approximately 1×10⁶ plaques from a human genomic library inλFixII available from Stratagene were screened by conventional methodswith a random primed EcoRI/BstXI fragment encompassing nucleotides485-1151 of the rat Gax cDNA. Two clones contained the second exon ofhuman Gax gene, having 182 base pairs. Using this coding information,the rest of the coding region was cloned by polymerase chain reactionmethods.

Reverse transcriptase and polymerase chain reaction techniques were usedto clone the 3′ end of the human cDNA. The template was whole human RNAisolated from human internal mammary artery isolated by TRI reagent fromMolecular Research Center, Inc. The following reagent concentrationswere used in the reverse transcriptase reaction: 1 μg of total internalmammary artery RNA; 50 mM Tris-HCl pH 8.5; 30 mM KCl; 8 mM MgCl₂; 1 mMDTT; 20 units RNAsin from Boehringer Mannheim; 1 mM each of dATP, dTTP,dGTP, and dCTP; 0.5 μg random hexamers from Boehringer Mannheim; and 40uof AMV reverse transcriptase from Boehringer Mannheim, in a total volumeof 20 μL. This was incubated for 1 hour at 42° C., heat inactivated, andthen stored at −80° C. before use. An initial amplification of 10% ofthe reverse transcriptase reaction was performed with just the senseoligonucleotide primer, known as “H2” and Ampliwax™ PCR Gem 100 beadsPerkin Elmer in a “hot start” procedure according to the directions ofthe manufacturer. The following reagent concentrations were used: 50 mMKCL; 10 mM Tris-HCl at pH 8.3; 1.5 mM MgCl₂; 1 mg/mL gelatin; 0.2 mMeach of DATP, dTTP, dGTP, and dCTP; 0.1 μM primer(s); and 2.5 units ofTaq polymerase from Boehringer Mannheim or Perkin Elmer in a volume of100 μL (these conditions were used thereafter unless noted). The cyclingprotocol was as follows: 94° C. for two minutes, then 30 cycles of 94°C. for 30 seconds, 45° C. for 1 minute, and 72° C. for 1 minute. Asecond amplification was then performed on 10% of the primary reactionproducts using the H2 primer and a degenerate antisense oligonucleotideprimer known as “P2B” against the carboxy terminal peptide. The cyclingparameters were: 94° C. for two minutes followed by 30 cycles of 94° C.for 30 seconds, 40° C. for 30 seconds, 50° C. for 1 minute and 72° C.for 1 minute. A product was observed of the correct size and followingpurification by Glass Fog from Bio101, on 2% Biogel agarose from Bio101was blunt sub-cloned into EcoRV digested BluescriptII SK+ vectors fromStratagene and sequenced to high resolution by Sequenase 2.0 fromuniversal primers from United States Biochemical. Five individual cloneswere sequenced to eliminate any spurious Taq polymerase errors.

The 5′ end of the human coding region was amplified using an anchoredpolymerase chain reaction kit, available under the tradename“5′-Amplifinder RACE” from Clonetech according to the manufacturer'sinstructions. This method uses single stranded RNA ligase to ligate ananchor oligonucleotide onto the 3′ end of appropriately primed firststrand cDNA. Templates used were either human heart polyA+ RNA obtainedfrom Clonetech or polyA+ RNA isolated from primary cultures of humanvascular smooth muscle cells obtained from Clonetics. The polyA+ RNAfrom cultured vascular smooth muscle cells was purified with RNAzol Bfrom Biotecx using batch chromatography on Oligo-dT latex beads fromQiagen. Both templates yielded amplified cDNAs and specific subcloneswere chosen solely by size. First strand RNA templates were prepared byeither specific priming or priming with random hexamers from BoehringerMannheim. In general, the specific primed templates yielded longerclones but could not be used for multiple step wise amplification of therest of the coding region.

Amplification from anchored templates using the sense anchor primer andappropriate antisense specific primers was accomplished using ampliwaxbeads from Perkin Elmer and “hot start” polymerase chain reaction usingthe same reaction conditions as above, but with 0.2 μM primers in atotal volume of 50 μL. The cycling protocol was as follows: 94° C. for 2minutes then 30 cycles of 94° C. 45 seconds, 60° C. 45 second, and 72°C. for 1.5 minutes, followed by a final extension of 72° C. for 10minutes. Following a primary amplification, aliquots (10-20%) of thereactions were run out on 2% Biogel agarose from Bio101 and sizeselected. After purification by glass fog from Bio101, 1-10% of theelutes were reamplified (2°), usually with a nested primer. Productswere observed at this point and purified by glass fog as before andsequenced directly using a thermal cycling kit from New England Biolabs.Once the products were confirmed they were sub-cloned as describedabove. Between 5 to 8 individual clones from each of three sequentialamplifications were sequenced to eliminate spurious Taq polymeraseerrors and appropriate clones chosen for the finished molecule. Asummary of the primer pairs sense/antisense used to amplify the completecoding region follows: Source of position Clone # Template 1° 2° 5′-3′ 6dN6 primed IMA H2 H2/P2B 699-941 whole RNA 23 H2R primed Heart AP/H2RAP/H3 231-698 poly A + RNA 117 dN6 primed VSMC AP/H6 AP/H6 119-230 polyA + RNA 131 dN6 primed VSMC AP/H6 AP/H7  1-118 poly A + RNA

Clones were pieced together 3′-5′ as follows: fragments 6 and 23 shareengineered BglII sites; fragments 23 and 117 share a native SfaNI site;fragment 117 has a native NcoI site which is compatible with anengineered BspHI site in fragment 131. Both engineered sites have asingle base change in the wobble base of leucine codons, as noted on thefinal sequence as shown in FIG. 3. Once assembled the molecule wasexcised by digestion with EcoRI and HindIII. The map in FIG. 4 shows themolecule and its orientation. TABLE 1 Primers Used to Amplify Human Gaxgene Primer Sequence 5′-3′ P2B TCA, IA (G/A), (G/A) TG, IGC, (G/A)TG,(T/C) TC H2 GCGCGC (AGATCT) CAC, TGA, AAG, ACA, GGT, AAA H2R TT, TAC,CTG, TCT, TTC, AGT, GAG H3 GCGCGC (AGATCT) AG, ATT, CAC, TGC, TAT, CTC,GTA H6 GCGCGTGCCCCCTCTGATG, CTG, GCT, GGC, AAA, CAT, GT H7 GCGCGC(TCTTGA) AGG, GCG, AGA, GAG, GAT, TGG, GA APCTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG Anchor GGAGACTTCCAAGGTCTTAGCTATCA(CTTAAG) CACEngineered enzyme sites are bracketed.The Gax Gene Maps to a Novel Locus on Chromosome 7 in the Human Genome

To determine the map location of Gax in the human genome, a 16.5kilobase pair fragment of the human genomic Gax gene in λFix II fromStratagene was purified with a Qiagen purification column according tothe directions of the manufacturer, and it was labeled with biotin11-dUTP by nick translation. Metaphase spreads of normal humanlymphocytes were prepared according to the methods of Fan, Y., Proc.Natl. Acad. Sci. (USA) Vol. 87, pp. 6223-6227 (1990). Fluorescence insitu hybridization and immunofluorescence detection were performedaccording to the methods of Pinkel, D., et. al., Proc. Natl. Acad. Sci.(USA) Vol. 83, pp. 2934-2938 (1986) and Testa, J. R., et al. Cytogenet.Cell. Genet. Vol. 60, pp. 247-249 (1992). Chromosome preparations werestained with diamidino-2-phenylindole and propidium iodide according toFan, Y. S., et. al., Proc. Natl. Acad. Sci. (USA) Vol. 87, pp. 6223-6227(1990).

Forty metaphase spreads were examined with a Zeiss Axiophot fluorescencemicroscope, and fluorescent signals were detected on the short arm ofchromosome 7 in 34 of these spreads. All signals were located atp15-->p22, with approximately 70% of the signals at 7p21. Based on thesedata, Gax is the only homeoprotein known to map to this locus.

Gax Gene Expression is Down-Regulated in Cultured Vascular Smooth MuscleCells Upon Mitogen Stimulation

It has been found that the Gax gene is expressed in quiescent vascularsmooth muscle cells. Since platelet derived growth factor hereinafteralso referred to as “PDGF” and other growth factors regulate vascularsmooth muscle proliferation and differentiation, differences in Gax geneexpression in response to PDGF and other mitogens such as fetal calfserum were examined in cultured vascular myocytes.

Cultures of rat smooth muscle cells were obtained from the media ofaortas isolated from adult male Sprague-Dawley rats. Cells were seededonto dishes in medium containing a 1:1 mixture of Dulbecco's modifiedEagle's medium and Ham's F12 and supplemented with 10% newborn calfserum. Once established, the cells were maintained at 37° C. in ahumidified atmosphere of 5% carbon dioxide, and subcultured within threedays after reaching confluence. Vascular smooth muscle cells werelabeled with monoclonal antibodies to smooth muscle α-actin from SigmaChemical Co. to verify identity.

The cultured cells were exposed to various mitogens as discussed below.The cells were then harvested and the total mRNA was extracted. Thetotal RNA from rat cultured cells was prepared by the guanidinethiocyanate method according to Chomcynzski, P., and N. Sacchi, (1987)“Single-step Method of RNA Isolation by Acid GuanidiniumThiocyanate-phenol-chloroform Extraction” Anal. Biochem. Vol. 162, pp.156-159, fractionated on 1.2% agarose gels containing formaldehyde, andblotted onto nylon membranes. The RNA from cultured cells was separatedon 30-cm gels for transcript size determination and on 10-cm gels forother studies. Hybridizations were carried out at 65° C. in buffercontaining 0.5 M sodium phosphate at pH 7.0, 7% sodium dodecyl sulfate,1 mM EDTA, and 1% bovine serum albumin, using a cDNA probe labeled byrandom priming consisting of a truncated Gax cDNA lacking the 5′ end andthe CAX repeat, where the X may be cytosine or guanine. Probes forHox-1.3 and Hox-1.4 consisted of the cDNAs isolated from the rat aortalibrary, and the probe for Hox-1.11 consisted of the DraI-EcoRI fragmentof its cDNA. The blots were washed with a final stringency of 0.1 to0.2×SSC-0.1% sodium dodecylsulfate at 65° C. After the probings with thehomeobox probes were complete, the blots were rehybridized with a probeto rat glyceraldehyde 3-phosphate dehydrogenase hereinafter alsoreferred to as “GAPDH,” to demonstrate message integrity. Gax mRNA andGAPDH mRNA were quantified with a Molecular Dynamics model 400SPhosphorImager to integrate bank intensities, or by scanningdensitometry of autoradiograms. In all quantitative comparisons of GaxmRNA levels between experimental groups, Gax mRNA levels were normalizedto the corresponding GAPDH level determined on the same blot, to accountfor differences in RNA loading.

Time Course of GAX Down-Regulation in Cultured Vascular Smooth MuscleCells

Rat vascular smooth muscle cells, grown to a greater than about 90%confluence, were placed in low-serum medium containing 0.5% calf serumfor 3 days, to induce quiescence. At this time, the medium was removedfrom the cells and replaced with fresh medium containing either 10%fetal calf serum or 10 ng/ml platelet derived growth factor from humanplatelets. The cells were then incubated for the various times in thepresence of either the fetal calf serum or the PDGF. As a control,quiescent cells were incubated with fresh serum-free medium alone. Thecells exposed to PDGF were harvested at 0.25, 0.5, 1, 2, and 4 hours,and the cellular RNA isolated. The cells exposed to human fetal calfserum were harvested at 4, 24, and 48 hours. The Gax and the Hox mRNAlevels were determined by Northern blot analysis. Typical results areshown in FIGS. 5A and 5B.

A rapid down-regulation, that is a reduction in the amount of Gax mRNA,occurred in the vascular smooth muscle cells when they were stimulatedwith either fetal calf serum or PDGF as shown in FIGS. 5A and 5B. Thedown-regulation ranged from 5- to nearly 20-fold, depending on themitogen used and the experiment. The down-regulation typically occurredwithin 2 hours after stimulation with fetal calf serum or PDGF, and wasmaximal at 4 hours. Gax mRNA transcript levels began to recoversignificantly by approximately 24 hours and approached baseline between24 and 48 hours after stimulation. The rate of recovery varied with themagnitude of the initial down-regulation and the individual cell culturepreparations. While PDGF isolated from human platelets caused a rapiddown-regulation of Gax, it had little or no effect on Hox-1.3 mRNAlevels. Neither fetal calf serum nor any of the three isoforms of PDGFshowed any effect on the transcript levels of Hox-1.3, Hox-1.4, orHox-1.11, homeobox genes which were also isolated from the vascularsmooth muscle library.

Magnitude of Gax Down-regulation Correlates With Potency of Mitogen

PDGF is a homodimer or heterodimer made of various combination of twochains, A and B. Thus, there are three isoforms of PDGF; PDGF-AA;PDGF-AB; and PDGF-BB; and they have differing potencies for stimulatingDNA synthesis in rat vascular smooth muscle cells. The PDGF-AA, PDGF-ABand PDGF-BB were compared for their effect on Gax expression. Quiescentrat vascular smooth muscle cell received 10 ng/ml of either PDGF-AA,PDGF-AB, PDGF-BB, or 10% fetal calf serum. After 0, 1, 2, 4 and 8 hoursthe cells were harvested and the Gax mRNA level determined. The resultsare shown in FIG. 6.

As shown in FIG. 6, PDGF-AA did not down-regulate Gax gene expression invascular smooth muscle cells, whereas the PDGF-AB and PDGF-BB isoforms,and the fetal calf serum reduced Gax gene expression approximately10-fold by 4 hours. The greatest down-regulation occurred with the fetalcalf serum followed by that with PDGF-BB and PDGF-AB.

To determine whether the extent of Gax gene down-regulation correlatedwith the potency of the mitogen used to stimulate the vascular smoothmuscle cells, the ability of the three PDGF isoforms and fetal calfserum to stimulate DNA synthesis was measured by ³H-thymidine uptake.Quiescent vascular smooth muscle cells were stimulated with one of thethree PDGF isoforms, at 10 mg/ml, or 10% fetal calf serum. Then 5 μCi/ml³H-thymidine was added to the cultures for 1 hour at various time pointsas shown in FIG. 7. The cells were harvested and the ³H-thymidine uptakewas measured. The results are shown in FIG. 7.

The PDGF-AA at 10 ng/ml, which was ineffective in causing Gax genedown-regulation, only weakly stimulated DNA synthesis as shown in FIG.7. PDGF-AB and PDGF-BB both stimulated cell proliferation as measured by³H-thymidine uptake at 15 hours. However, the fetal calf serum which wasmost effective at down regulating Gax gene expression, was also the mosteffective mitogen, that is it demonstrated the greatest ³H-thymidineuptake.

Down-Regulation of the Gax Gene is Dependent on the Dose of the Mitogen

Dose-response experiments were conducted by stimulating quiescentvascular smooth muscle cells with either PDGF-AB, PDGF-BB or fetal calfserum at varying doses as shown in FIGS. 8 and 9. The effects on GaxmRNA levels were measured at 4 hours after mitogen stimulation. Theresults are shown in FIGS. 8 and 9.

As shown in FIG. 8, the dose response curves reveal that the 50%effective dose for Gax gene down-regulation 4 hours after PDGF-ABstimulation is between 4 and 8 ng/ml. The 50% effective dose for Gaxgene down regulation 4 hours after PDGF-BB stimulation is between 2 and5 ng/ml. The 50% effective dose for Gax down regulation 4 hours afterfetal calf serum is approximately 1%, as shown in FIG. 9. Furthermore,10% fetal calf serum suppresses Gax mRNA levels nearly 20-fold at 4hours, an effect larger than that of a maximal stimulatory dose ofPDGF-BB (30 ng/ml), which has a 10-fold effect, or of PDGF-AB, which hasa less than 8-fold effect, as shown in FIG. 6. Thus, the down-regulationof Gax gene induced by either fetal calf serum or the different isoformsof PDGF correlates well with their abilities to stimulate DNA synthesisas measured by ³H-thymidine uptake.

The Gax gene down-regulation is sensitive to low levels of mitogenstimulation, which cause a significant decrease in Gax mRNA levels. Asshown in FIG. 9, stimulation of quiescent rat vascular smooth musclecells with 1% fetal calf serum caused a 40% decrease in Gax mRNA levelsafter 4 hours. However, such stimulation increased ³H-thymidine uptakeless than two-fold over that observed in quiescent vascular smoothmuscle cells (data not shown). Treatment with PDGF-BB at doses as low as2 ng/ml, also caused a detectable decrease in the Gax mRNA level.

Gax Expression is Up-Regulated or Induced when Synchronously GrowingCells Are Deprived of Serum

Sparsely plated vascular smooth muscle cells were grown in a mediumcontaining 20% fetal calf serum, and then placed into serum free medium.The RNA was harvested at various times from 0 to 25 hours. The totalmRNA was extracted and subjected to Northern Blot Analysis, then themRNA transcript of Gax was quantified.

As shown in FIG. 10, the expression of the Gax gene was induced fivefoldin vascular smooth muscle cells within 24 hours after the rapidlygrowing cells were placed in the serum-free medium. Thus, expression ofGax gene is regulated by the growth state of the cell, and itsdown-regulation is a prominent feature of the G₀/G₁ transition in thesecells.

Gax Protein Inhibits Mitogen-Induced S Phase Entry in Vascular SmoothMuscle Cells

Production of Recombinant Proteins

To determine whether Gax gene exerts a negative control on cell growthin vascular smooth muscle cells, Gax gene was expressed as a glutathioneS-transferase (hereinafter also referred to as “GST”) fusion protein inbacteria and microinjected it into quiescent vascular smooth musclecells. To determine the effect of the Gax protein on serum-induced cellproliferation, the effect of GST-Gax protein was compared to the effectof known protein regulators of cell growth.

To produce the Gax protein evaluated herein, the cDNA coding regions forGax was fused in frame to the pGEX-2T expression vector obtained fromPharmacia Biotechnology, and then expressed in E. coli. Specifically,GST-Gax was produced according to the following procedure: the codingregion of Gax cDNA spanning from nucleotides 200-1108 was amplified bypolymerase chain reaction methods using the following primers:5′ GCGCGCGTCGACGAACACCCCCTCTTTGGC 3′ and5′ GCGCGCAAGCTTTCATAAGTGTGCGTGCTC 3′

The resulting DNA was digested with SalI and HindIII restriction enzymesand cloned into SalI and HindIII sites in the polylinker of pGEM3-1T invitro transcription translation vector described in Patel R. C. and SenG. C. (1992) “Identification of the Double-stranded RNA-binding Domainin the Human Interferon-inducible Protein Kinase,” J. Biol. Chem. Vol.267; pp. 7671-7676. The BamHI to NaeI fragment of pGEM3-1T containingthe Gax coding region was then sub-cloned into the same sites ofpGEX-2T. The pGEX-2T vector with the YY1 cDNA, used to produce GST-YY1,was from Thomas Shenk at Princeton University.

The resultant glutathione S-transferase fusion proteins were purified byaffinity chromatography on glutathione-agarose beads. E. coli XL1-bluecells were then transformed with the appropriate plasmid and were grownto a density of 0.6-0.8 A₆₀₀ and induced with 0.5 mMisopropyl-B-D-thiogalectopyrenoside for 2 hours. The cells wereharvested and lysed by ultrasonic vibration in phosphate buffered salinecontaining 1% triton x-100, 1 mM PMSF and 5 μg/ml aprotinin. The lysatewas centrifuged at 15,000×g and the supernatant was collected. Thesupernatant was bound to the glutathione sepharose from Pharmacia (0.5ml of resin per 100 ml of bacterial culture) for 2 hours on a rotator at25 rpm. The slurry was pelleted by centrifugation at 1000×g for 2minutes, then washed twice with complete lysis buffer then washed twicewith lysis buffer lacking triton x-100. The bound protein was eluatedfor 30 minutes with phosphate buffered saline containing 10 mM reducedglutathione, from Sigma Chemical Company, 40 mM DTT and 150 mM NaCl.Purity of the GST-Gax protein was greater than 90% as determined bySDS-PAGE gels stained with Coomassie blue.

To produce recombinant MHox, its cDNA was fused in frame to the pQE-9 E.coli expression vector obtained from Qiagen, Inc., Chatsworth, Calif.,then expressed in bacteria, and purified by adsorption to a nickelcolumn.

For microinjection, proteins were concentrated in a buffer containing of20 mM Tris, 40 mM KCl, 0.1 mM EDTA, 1 mM β-mercaptoethanol, and 2%glycerol using Centricon-30 from Amicon microconcentrators. Concentratedproteins were stored in this buffer in aliquots at −80° C.

Microinjection and Cell Culture Methods

Microinjections were performed using a semiautomatic microinjectionsystem from Eppendorf Inc. in conjunction with a Nikon Diaphot phasecontrast microscope. According to Peperkole, R., et al. (1988) Proc.Natl. Acad. Sci. USA Vol. 85, pp. 6758-6752, The injection pressure wasset at 70-200 hPa and the injection time was 0.3 to 0.6 seconds.

After injection, cells were stimulated 24 hours with medium containing10% fetal calf serum, and the incorporation of 5′-bromo-2′-deoxyuridine,hereinafter also referred to as “BrdU” was measured with a cellproliferation kit according to the directions of its manufacturer,Amersham. When fetal calf serum-stimulated BrdU labeling was determined,BrdU was included for 24 hours with the medium used to stimulate thecells. Where the ability of microinjected proteins to stimulate growthin serum-poor medium was measured, cells were incubated 24 hours in thesame low serum medium used to induce quiescence, but supplemented withBrdU. After labeling, the cells were fixed with acid-ethanol, and thepercentage of nuclei positive for BrdU uptake was determined forprotein-injected and buffer-injected cells. The percent of cell growthinhibition was calculated according to the following formula:

where IL represents the number of injected labeling positive for BrdU;IT, the total number of injected cells; CL the number ofcontrol-injected cells labeling with BrdU; CT, the total number ofcontrol-injected cells counted. With this equation, inhibition ofmitogen-induced entry into S phase is represented by a positive numberand stimulation of cell growth is represented by a negative number.

Evaluation of Gax Protein

To determine if the Gax protein inhibits the entry of mitogen stimulatedvascular smooth muscle cells into S-phase, the effect of the Gax proteinwas compared to proteins known to effect cell proliferation, and tocontrol proteins. Such comparison proteins include a neutralizingantibody against ras, “Y13-259,” which is highly effective in blocking Sphase entry when microinjected into NIH3T3 cells; the transcriptionfactor MHox, a homeodomain protein unlikely to have an inhibitory effecton cell proliferation; and YY1, a zinc finger transcription factorunlikely to have a negative effect on cell growth.

Quiescent rat vascular smooth muscle cells were microinjected witheither 0.6 mg/ml GST-Gax protein; 1.6 mg/ml MHox; 1.2 mg/ml YY1; 8 mg/mlY13-259; 2 mg/ml GST alone; or 8 mg/ml mouse anti-human IgG. The cellswere then stimulated for 24 hours with 10% fetal calf serum in mediumcontaining BrdU. After 24 hours, the fraction of nuclei labeling withBrdU was determined and percentage inhibition of S-phase entrycalculated. The results are summarized in Table 2. TABLE 2 Effect ofMicroinjected Proteins on the Serum-induced Proliferation of VascularSmooth Muscle Cells Mean % Inhibition of Total Number FCS-stimulatedNumber of of Cells Growth ± Treatment Experiments Examined StandardError Antibody Y13-259 2 328 60.8 ± 3.9 Mouse anti-human IgG 3 330 −3.4± 4.5 GST-Gax 15 2943 42.7 ± 3.3 MHox 2 236 −5.3 ± 9.3 GST-YY1 5 306 0.0 ± 12.2 GST 7 1144 −2.6 ± 2.1FCS—fetal calf serumBrdU labeling of quiescent vascular smooth muscle cells was 10.1 ± 1.2%(N = 12, total number of cells counted = 2659); for uninjectedFCS-stimulated vascular smooth muscle cells, 54.8 ± 2.4% (N = 27, totalnumber of cells counted = 4282); and for sham-injected FCS-stimulatedcells, 49.6 ± 2.5% (N = 27, total number of cells injected = 3401).

As shown in Table 2, the GST-Gax protein inhibited vascular smoothmuscle cell entry into S-phase by 42.7%. The GST Gax protein effect onmitogen-stimulated entry into S phase is specific. The other injectedproteins GST, YY1, MHox and the mouse anti-human IgG failed to inhibitvascular smooth muscle cell growth. In comparison to the GST-Gaxprotein, the antibody Y13-259, as anticipated, significantly decreasedmitogen-induced cell proliferation. Vascular smooth muscle cellsmicroinjected with Y13259 demonstrated a 61±4% decrease in cell entryinto S-phase

Gax Protein Inhibits Vascular Smooth Muscle Cell Proliferation in aDose-Dependent Manner.

To determine the concentration of microinjected GST-Gax required toinhibit vascular smooth muscle cell growth, solutions containingdifferent concentrations of GST-Gax protein were microinjected intoquiescent vascular smooth muscle cell and the effects onmitogen-stimulated entry into S phase examined. Specifically, vascularsmooth muscle cells were rendered quiescent by incubation in mediumcontaining 0.5% calf serum for three days. The cells were microinjectedwith varying concentrations of GST-Gax, and stimulated with 10% fetalcalf serum, and labeled with BrdU. After 24 hours, the percentageinhibition of cell proliferation was determined. Each data pointrepresents the mean±standard error of 3-5 experiments in which 100-200cells per experimental group were injected.

As shown in FIG. 11, the cellular growth inhibition by the GST-Gaxprotein is dose dependent. Little or no growth inhibition was observedwhen 0.2 mg/ml GST-Gax protein was injected. The maximal growthinhibition was obtained with approximately 0.5 mg/ml of the GST-Gaxprotein.

Gax Inhibits Proliferation of Several Cell Types

To determine whether the GST-Gax protein inhibits growth in other cellstypes, the GST-Gax protein was microinjected into quiescentSV40-transformed vascular smooth muscle cells, BALBc3T3 cells, NIH3T3cells, human vascular smooth muscle cells, and human fibroblasts. TheSV40 transformed cell line was derived from rat vascular smooth musclecells transformed with the SV40 large T antigen. These cells, whileimmortalized, retain many differentiated characteristics ofuntransformed vascular smooth muscle cells. The cells were microinjectedwith either 0.6 mg/ml GST-Gax protein or 2 mg/ml GST were thenstimulated with 10% fetal calf serum, and labeled for 24 hours withBrdU. The results are shown in Table 3. TABLE 3 EFFECT OF MICROINJECTEDGST-GAX PROTEIN ON CELL PROLIFERATION IN DIFFERENT CELL TYPES Mean &Range Inhibition Mitotic Number Number of of FCS- Index in GST-GAX ofCells Stimulated Response Cell type protein Experiments Examined Growthto FCS SV40- Yes 4 448 27.2 ± 2.0 — transformed VSMC SV40- No N/A 0.60 ±0.02 transformed VSMC BALB/c Yes 4 464  30.5 ± 10.9 — 3T3 cells BALB/cNo N/A 0.64 ± 0.03 3T3 cells NIH3T3 cells Yes 4 420 23.2 ± 1.8 — NIH3T3cells No N/A 0.70 ± 0.02 Human VSMC Yes 3 506 46.6 ± 8.1 — Human VSMC NoN/A 0.33 ± 0.02 Human Yes 3 336 44.5 ± 2.1 — fibroblasts Human No N/A0.36 ± 0.01 fibroblastsFCS—fetal calf serumVSMC—vascular smooth muscle cells

SV40—transformed vascular smooth muscle cell proliferation was inhibitedby GST-Gax protein, as shown in Table 3. The GST-Gax protein alsoinhibited the proliferation of fibroblast cell lines NIH3T3 and BALB/c3T3. GST-Gax protein also inhibited the proliferation of human cells,specifically human vascular smooth muscle cells and human foreskinfibroblasts. These results indicate that Gax action is not celltype-specific, although there are differences in the extent inhibitionamong the different cell types. The Among the human cells, the GST-Gaxprotein exhibits maximal inhibition in vascular smooth muscle cells, thecell type in which the Gax gene is normally expressed. Similarly amongthe rat cells, the GST-Gax protein exhibits maximal inhibition invascular smooth muscle cells, the cell type in which the Gax gene isnormally expressed.

An Oncogenic Ras Protein Can Reverse Growth Inhibition Caused by the GaxProtein

To characterize the mechanism of the growth inhibition conferred by theGST-Gax protein, the effects of GST-Gax protein and the transformingoncoprotein, the ras mutant Ras(Leu-61) were compared by microinjectingthese proteins into rat vascular smooth muscle cells. A solutioncontaining both 0.5 mg/ml GST-Gax protein and 0.5 mg/ml Ras(Leu-61) wasmicroinjected into quiescent vascular smooth muscle cells. Forcomparison, other vascular smooth muscle cells received either 0.5 mg/mlGST-Gax protein or 0.5 mg/ml Ras(Leu-61) or 0.5 mg/ml GST. The injectedcells were then incubated for 24 hours with medium containing 10% fetalcalf serum and BrdU. The results are shown in FIG. 12.

As shown in FIG. 12, when Ras(Leu-61) alone was injected, there was anincrease in BrdU-labeling as compared to both control-injected cells. Incells injected with GST-Gax protein, growth was inhibited 39%. When theGST-Gax protein and Ras(Leu-61) were coinjected in the cells, theRas(Leu-61) reversed the growth inhibitory effects of the GST-Gaxprotein, and the percentage of cells staining positive for BrdU in cellsreceiving both the Ras(Leu-61) and GST-Gax protein were nearly identicalto that observed in cells receiving just the Ras(Leu-61). Thus, the Rasoncoprotein completely reversed the effect of the GST-Gax proteinestablishing that the presence of GST-Gax protein is not toxic to cells.

the Gax Protein Inhibits Cell Growth when Microinjected Before the G1 toS Boundary

To determine the point in the cell cycle when the Gax gene exerts itsgrowth inhibitory effects, the time of S phase onset was determined inrat vascular smooth muscle cells. The vascular smooth muscle cells werestimulated with 10% fetal calf serum and pulse labeled with 10 mCi/ml³H-thymidine for one hour at different times after stimulation. Seraratecultures of the cells were microinjected with GST-Gax protein at varioustimes after receiving 10% fetal calf serum and labeled with BrdU between10 and 24 hours after receiving the fetal calf serum. Percent inhibitionof S-phase entry was determined at each time point. The results areshown in FIG. 13.

As shown in FIG. 13, S phase onset, indicated by the uptake of³H-thymidine, occured at approximately 16-18 hours after mitogenstimulation. GST-Gax protein significantly inhibited vascular smoothmuscle cell entry into the S phase when microinjected at any time fromstimulation up until approximately 12 hours. However, GST-Gax proteinwas ineffective when injected at 15 hours. Thus it appears that the Gaxgene inhibits a critical step in cell cycle progression prior to theG₁/S boundary; perhaps before the restriction point in G₁ whereeukaryotic cells are irreversibly committed to entering the S phase.

Gax Gene Expression is Rapidly Down Regulated In Vivo Upon Acute BloodVessel Injury

The Gax gene expression in normal blood vessels and in injured bloodvessels was compared to determine whether Gax gene down-regulationoccurs in response to injury-induced smooth muscle cell proliferation invivo. Adult male Sprague-Dawley rats were subject to acute vessel injuryby balloon de-endothelialization in the carotid arteries according tothe methods of Majesky, M. W., et al. J. Cell. Biol. (1990) Vol. 111,pp. 2149-2158. The expression levels of Gax, that is, the mRNA levels,were assessed relative to that of glyceraldehyde 3-phosphatedehydrogenase (hereinafter also referred to as “G3”) by a quantitativepolymerase chain reaction. At various times following balloonde-endothelialization the rats were sacrificed and the total RNA wasisolated from the vascular smooth muscle tissues using the TRI reagentfrom Molecular Research Center, Inc. The cDNA was synthesized from theextracted RNA with MMLV reverse transcriptase from Bethesda ResearchLabs. Aliquots of the cDNA pools were subjected to polymerase chainreaction amplification with AmpliTaq DNA polymerase from Perkin-Elmer inthe presence of α32P-dCTP with the following cycle conditions: 94° C.for 20 seconds, 55° C. for 20 seconds, and 72° C. for 20 seconds. Thefinal cycle had an elongation step at 72° C. for 5 minutes. The primersfor the rat Gax amplification were: 5′-CCCGCGCGGCTTTTACATTAGGAGT-3′ and5′-GCTGGCAAACATGCCCTCCTCATTG-3′. The primers for the rat G3 gene were5′-TGATGGCATGGACTGTGGTCATGA-3′ and 5′-TGATGGCATGGACTGTGGTCATGA-3′. TheGax cDNA was amplified for 30 cycles, and G3 was amplified for 25 cyclesin the same reaction vessels. The amount of a radioactive labelincorporated into the amplified cDNA and G3 fragments was determined bysubjecting the fragments to electrophoresis on a 1% agarose gel, thenexcising the bands and liquid scintillation counting. Since the mRNAlevels of glyceraldehyde 3-phosphate dehydrogenase remain relativelyconstant following this procedure (see J. M. Miano et al. 1990, Am. J.Path. 137, 761-765), the ratio of radiolabel incorporation into theGax-derived amplified bands and the G3-derived amplified bands correctsfor differences arising from the efficiency of RNA extraction from thedifferent animals, and it provides a measure of Gax mRNA levels in thenormal and injured vascular tissues. These ratios are plotted in FIG.14.

As shown in FIG. 14, the Gax mRNA expression was down-regulated inresponse to acute vessel injury by as much as a factor of 20. Thisdown-regulation was rapid and appeared complete by 2 hours, the firsttime-point following the de-endothelialization procedure. Collectively,these data corroborate the Gax gene down-regulation in cultures ofvascular smooth muscle cells following growth factor stimulation.Further, these data show that Gax gene expression is an early marker ofthe cell cycle activity associated with the initiation of vascularrestenosis, and they indicate that Gax has a regulatory role followingblood vessel injury.

The present invention includes: the DNA sequences encoding a protein, orportion thereof, which inhibits vascular smooth muscle cellproliferation; the messenger RNA transcript of such DNA sequence; and anisolated protein which inhibits vascular smooth muscle cell growth.

For example, the DNA sequences include: DNA molecules which, but for thedegeneracy of the genetic code would hybridize to DNA encoding the Gaxprotein, thus the degenerate DNA which encodes the Gax protein; DNAstrands complementary to DNA sequences encoding the Gax protein orportions thereof including DNA in FIGS. 1 and 3 or portions thereof;heterologous DNA having substantial sequence homology to the DNAencoding the Gax protein, including the DNA sequences in FIGS. 1 and 3or portions thereof.

The isolated protein includes, for example, portions of the Gax protein;the Gax protein of animals other than rat and human; and proteins orportions thereof having substantially the same amino acid sequence asshown in FIGS. 1 and 3 or portions thereof.

1-6. (canceled)
 7. An isolated protein which inhibits vascular smoothmuscle cell growth, wherein said protein comprises the amino acidsequence shown in FIG. 1, SEQ ID NO. ______ or the amino acid sequenceshown in FIG. 3, SEQ ID NO. ______.
 8. The protein of claim 7 having amolecular weight of from about 30 kDA to about 36 kDa.
 9. An isolatedprotein which inhibits vascular smooth muscle cell proliferation,wherein said protein is encoded by a polynucleotide comprising thefollowing fragments: (a) a fragment of clone 6 comprising nucleotides699 to 941 of said polynucleotide: (b) a fragment of clone 23 comprisingnucleotides 231 to 698 of said polynucleotide; (c) a fragment of clone117 comprising nucleotides 119 to 230 of said polynucleotide, and (d) afragment of clone 131 comprising nucleotides 1 to 118 of saidpolynucleotide.
 10. (canceled)
 11. The protein of claim 7, comprisingthe amino acid sequence as shown in FIG.
 1. 12. The protein of claim 7,further comprising glutathione S-transferase. 13-32. (canceled)